Perinatal exposure to smoking and parenteral nicotine has been linked to a high risk of asthma, not just in the exposed offspring, but also transgenerationally in progeny who never encounter any form of nicotine at least as far as the F3 generation. Use of electronic-cigarettes (e-cigs) by pregnant women has been increasing, largely the result of the perception that vaping is relatively safe. This perception is suspected to be erroneous, but few objective studies have evaluated the adverse health effects of vaping. Our investigation will address the effects of vaping and flavorings used in vaping on development of asthma in progeny of mice exposed to e-cigs during pregnancy. We also ask whether this risk is transgenerationally inherited, and how heritability might occur. Germ cells are the only cells that are passed from one generation to the next, and have been shown to be exquisitely sensitive to environmental insults. Based on these considerations and on our preliminary data, we hypothesize that exposure to vaping disrupts the epigenetic machinery in germ cells, causing alterations of epigenetic marks across the genome that leads to a transgenerationally heritable asthma phenotype. Using established models, we will test this hypothesis in vivo in mice by determining whether e-cigs increase risk of asthma in: 1) offspring of pregnant F0 mothers and 2) offspring of F1 and F2 mothers who were nave to e-cig or nicotine exposure. We will test whether vaporized nicotine (AIM 1A) and flavorings (AIM 1B) have independent effects on transgenerational asthma risk, whether these risks are additive and whether they are exacerbated on allergen sensitization (AIM 1C). Nicotine-induced asthma is associated with a marked alteration of lung fibroblast phenotype, and we hypothesize that these alterations are reversible once the epigenome is restored towards normal. Hence, in AIM 2, we will assess the effects of nicotine and e-cig flavorings on viability (AIM 2A) and epigenetic memory (AIM 2B) of germ cells. Using multi-dimensional -omics tools (the Mergeomics platform) developed by our Co-I Xia Yang (UCLA), we will analyze data generated on molecular pathways across different data sets to determine how germ cell epigenetics are impacted by e-cigs with or without flavorings (AIM 2C). Finally, studies proposed in AIM 2D will attempt to reverse epigenetic changes in fibroblasts isolated from F3 progeny produced by e-cigs using RNAi. Dependent variables will include expression of molecular phenotypic markers that are the hallmarks of the asthmatic phenotype, and functional characteristics of fibroblasts isolated from F3 progeny, compared with appropriate controls. We anticipate that studies in Aim 2 will show reproducible alteration of the phenotype of lung fibroblasts that have differentiated from germ cells in mice that have either: 1) been directly exposed to e-cigs; or 2) never been exposed to nicotine in any form, but inherited the effects of ancestral exposure to e-cigs. These studies will importantly advance our mechanistic knowledge of how vaping might lead to persistent, transgenerationally-inherited risk of asthma in offspring. Further, our studies will help inform regulatory policies concerning exposure to e-cig nicotine and flavorings from vaping.